ProjectSummary/Abstract The long-term goal of my research is to understand the regulatory networks involved in epithelial homeostasisandskincarcinogenesis,aswellashumandiseasemorebroadly.Skincarcinogenesisis causedbythemalfunctionofnetworkscontrollingdifferentiationandproliferationinskincells.Avariety oftranscriptionalregulatorsofepidermaldifferentiationhavebeendescribed,includingmanyfactorsalso functionalinnon-skindisorders.Incontrast,onlyasmallnumberofnon-codingRNAshavebeenfound to be essential to epidermal proliferation or differentiation. In human skin, the long non-coding RNA (lncRNA) terminal differentiation-induced non-coding RNA (TINCR) is required for proper keratinocyte differentiation, and the lncRNA anti-differentiation non-coding RNA (ANCR) is essential to repress premature differentiation in keratinocytes. Both ANCR and TINCR affect carcinogenesis. The mechanismsbehindtheclearphenotypiceffectsofthesetwolncRNAs,bothdiscoveredintheKhavari lab, are not understood in detail. My proposal investigates the mechanisms behind the control of epidermal differentiation and proliferation by the lncRNAs ANCR and TINCR. Specifically, we will test the hypothesis that ANCR prevents ectopic differentiation by recruiting histone modifying enzymes to genomiclocibydeterminingprotein,RNAandDNAfactorsboundbyANCRusingthemethodsRNA- proteinmicroarray(RNA-PMA),RNA-interactomeanalysis(RIA-seq),andChromatinIsolationbyRNA Purification (ChIRP). While this aim will test a specific hypothesis, it is also aimed provide unbiased, genome-wide data on any potential ANCR mechanism. ANCR-interacting partners will be tested for functionalityinRNAiexperiments.TINCRisknowntobindtheRNA-bindingproteinStaufenandtoshare withStaufenasubsetofitstargetRNAs.TINCRbindstargetRNAspartiallythroughWatson-Crickbase- pairing.HowTINCRandStaufenaffecteachother?sRNAinteractionsisunknown,andwehypothesize it represents the interesting case of a programmable lncRNA-protein complex. We will determine the effectofTINCRonStaufen-RNAinteractionsusinganimprovedmethodofinfraredcrosslinkingfollowed byimmunopurification(irCLIP+),aswellastheeffectofStaufenonTINCR-RNAinteractionsusingRIA- seq,andwewilluseourresultstoconstructandtestmodelsofRNAtargetselectionbyaprotein-lncRNA complex.Altogether,thisproposalwillinvestigatethemechanismoflncRNA-mediatedcontrolofskincell differentiationandproliferation,aswellascarcinogenesis.